Retroviral expression in embryonic stem cells and hematopoietic stem cells

Achieving long-term retroviral expression in primary cells has been problem atic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the develo pment of a mouse stem cell virus (MSCV) long terminal repeat-based retrovir al vector that is expressed in both embryonic stem (ES) cells and hematopoi etic stem (HS) cells. Infected HS cells and their differentiated descendant s maintained long-term and stable retroviral expression after serial adopti ve transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP exp ression of integrated proviruses was maintained after in vitro differentiat ion of infected ES cells. Long-term passage of infected ES cells resulted i n methylation-mediated silencing, while short-term expression was methylati on independent. Tissues of transgenic animals, which we derived from ES cel ls carrying the MSCV-based provirus, did not express GFP. However, treatmen t with the demethylating agent 5-azadeoxycytidine reactivated the silent pr ovirus, demonstrating that DNA methylation is involved in the maintenance o f retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-inde pendent mechanisms.